Amplification of nucleic acids

ABSTRACT

Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b) using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands to as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to shuttling, a plurality of times, between an upper temperature and a lower temperature during the amplification process of steps (b)-(d), wherein at the upper temperature, one of said polymerase or nicking enzyme is more active than the other of said enzymes, such that there is a disparity in the activity of the enzymes, and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/956,552, filed on Jun. 19, 2020, which is the National Stageapplication under 35 U.S.C. § 371 of PCT International Application No.PCT/GB/2019/050005, filed on Jan. 2, 2019, which claims priority to andthe benefit of United Kingdom patent application No. 1800109.9, filedJan. 4, 2018, the entire disclosure of each of which is incorporatedherein by reference for all purposes.

REFERENCE TO A SEQUENCE LISTING XML

This application contains a Sequence Listing which has been submittedelectronically in XML format. The Sequence Listing XML is incorporatedherein by reference. Said XML file, created on Oct. 13, 2023, is namedLDX-019C1_SL.xml and is 17,413 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a method of amplifying a nucleic acidmolecule, especially in a quantitative manner.

BACKGROUND OF THE INVENTION

The polymerase chain reaction (PCR) is well-known and a standardtechnique used to amplify nucleic acid molecules. The amplified productsof the PCR are detected at the end of the reaction. The amount ofproduct tends to reach a plateau level, which does not increase if thereaction mixture is left longer. As a result, in conventional PCR theamount of product does not necessarily correlate with the concentrationof amplification target sequence present in the mixture at the outset.

In order to obtain quantitative data, quantitative PCR (“qPCR”) isperformed, in which the amount of amplification product produced ismonitored or detected in real time (hence qPCR is also referred to as“Real-Time PCR” or even “RT-PCR”, although this latter abbreviation isunhelpful as it can be confused with Reverse Transcriptase-PCR), whilstthe reaction is still actively amplifying the target sequence.

Typically, amplified nucleic acid is detected by its interaction with alabel entity (usually the label is a fluorophore). This interaction maybe non-specific (i.e. the label entity binds to essentially anydouble-stranded DNA molecule) or specific (i.e. the label entityinteracts in a nucleotide-sequence dependent manner preferentially witha specific nucleic acid sequence present in the desired amplificationproduct). An example of a non-specific label entity is the dye SYBR®Green. A specific label entity (e.g. a labelled probe molecule) mightbe, for example, a “molecular beacon”, which fluoresces when itundergoes a conformational change induced by hybridisation to a targetsequence.

Thus, monitoring the level of fluorescence observed in real time, duringthe PCR, allows the generation of quantitative data, in which the amountof amplification product (as measured by detection of fluorescence, forexample) correlates with the concentration of the amplification targetmolecule in the sample.

qPCR as described in U.S. Pat. No. 6,814,943 utilises temperature rangesfor cycling. Typically for qPCR the following procedure is undertaken:denaturation around 95° C., annealing around 55° C., extension around70° C. These are large temperature changes (about 40° C. differencebetween maximum and minimum temperatures). As a result qPCR, like“normal” non-quantitative PCR, requires the use of relativelysophisticated thermal cycling apparatus. Thus, whilst qPCR is highlyuseful in a research context (e.g. quantification of gene expression),it is not readily applicable to point-of care (“PoC”) diagnostic testsand the like.

Many nucleic acid amplification techniques have been devised, which areperformed isothermally, in order to avoid the need for thermal cycling.A non-exhaustive list of such amplification techniques includes: signalmediated amplification of RNA technology (“SMART”; WO 99/037805);nucleic acid sequence based amplification (“NASBA” Compton 1991 Nature350, 91-92); rolling circle amplification (“RCA” e.g. see Lizardi etal., 1998 Nature Genetics 19, 225-232); loop-mediated amplification(“LAMP” see Notomi et al., 2000 Nucl. Acids Res. 28, (12) e63);recombinase polymerase amplification (“RPA” see Piepenberg et al., 2006PLoS Biology 4 (7) e204); strand displacement amplification (“SDA”);helicase-dependent amplification (“HDA” Vincent et al., 2004 EMBO Rep.5, 795-800): transcription mediated amplification (“TMA”), single primerisothermal amplification (“SPIA” see Kurn et al., 2005 ClinicalChemistry 51, 1973-81); self-sustained sequence replication (“3SR”); andnicking enzyme amplification reaction (“NEAR”).

SDA is a technique (disclosed by Walker et al., 1992 Nucl. Acids Res.20, 1691-1696) which involves the use of a pair of primers comprising atarget-complementary portion and, 5′ of the target-complementaryportion, a recognition and cutting site for an endonuclease. The primershybridise to respective complementary single stranded target molecules.The 3′ end of the target strands are extended using a reaction mixincluding a DNA polymerase and at least one modified nucleotidetriphosphate, using the primer as template (and likewise, the 3′ ends ofthe primers are extended using the target as template).

The extension of the target strands generates a double strandedrecognition site for the endonuclease. However, because the target isextended using a modified triphosphate, the endonuclease does not cleaveboth strands but instead makes a single stranded nick in the primer. The3′ ends at the nicks are then extended by the DNA polymerase (typicallyKlenow fragment of DNA polymerase I, which lacks an exonucleaseactivity). As the nicked primers are extended, they displace theinitially-produced extension product. The displaced product is then freeto hybridise to the opposite primer, since it essentially replicates thesequence of the target for the opposite primer. In this way, exponentialamplification of both strands of the target sequence is achieved.

The amplification stage of the SDA process is essentiallyisothermal—typically performed at 37° C.—the optimum temperature for theendonuclease and the polymerase. However, before reaching theamplification stage it is necessary to completely dissociate the doublestranded target into its constituent single strands, in order to allowthe pair of primers to hybridise to their complementary target strands.

This dissociation, or “melting” is normally accomplished by heating thedouble stranded target to a high temperature—usually about 90° C.—inorder to break the hydrogen bonds between the two strands of the target.The reaction mix is then cooled to allow the addition of the enzymeswhich are necessary for the amplification reaction. Because of the hightemperature used to generate the single stranded targets, the SDAtechnique is not ideally suited to a PoC context.

U.S. Pat. No. 6,191,267 discloses the cloning and expression of N.BstNBInicking enzyme and its use in SDA, in place of restriction endonucleasesand modified triphosphates.

Another amplification technique, which is similar to SDA, is NickingEnzyme Amplification Reaction (or “NEAR”).

In ‘NEAR’ (e.g. as disclosed in US2009/0017453 and EP 2,181,196),forward and reverse primers (referred to in US 2009/0017453 and EP2,181,196 as “templates”) hybridise to respective strands of a doublestranded target and are extended. Further copies of the forward andreverse primers (present in excess) hybridise to the extension productof the opposite primer and are themselves extended, creating an“amplification duplex”. Each amplification duplex so formed comprises anicking site towards the 5′ end of each strand, which is nicked by anicking enzyme, allowing the synthesis of further extension products.The previously synthesised extension products can meanwhile hybridisewith further copies of the complementary primers, causing the primers tobe extended and thereby creating further copies of the “amplificationduplex”. In this way, exponential amplification can be achieved.

NEAR differs from SDA, in particular, in that no initial thermaldissociation step is required. The initial primer/target hybridisationevent needed to trigger the amplification process takes place whilst thetarget is still substantially double stranded: it is thought that theinitial primer/target hybridisation takes advantage of localiseddissociation of the target strands—a phenomenon known as “breathing”(see Alexandrov et al., 2012 Nucl. Acids Res. and review by Von Hippelet al., 2013 Biopolymers 99 (12), 923-954). Breathing is the localisedand transient loosening of the base pairing between strands of DNA. Themelting temperature (Tm) of the initial primer/target heteroduplex istypically much lower than the reaction temperature, so the tendency isfor the primer to dissociate, but transient hybridisation lasts longenough for the polymerase to extend the primer, which increases the Tmof the heteroduplex, and stabilises it.

The amplification stage in NEAR is performed isothermally, at a constanttemperature. Indeed, it is conventional to perform both the initialtarget/primer hybridisation, and the subsequent amplification rounds, atthe same constant temperature, usually in the range 54 to 56° C.

Avoiding the need for thermal cycling means that NEAR is potentiallymore useful than PCR in PoC contexts. In addition, synthesis ofsignificant amounts of amplification product, even when starting from avery low copy number of target molecules (e.g. as few as 10 doublestranded target molecules), can be achieved.

WO 2011/030145 (Enigma Diagnostics Limited) discloses the idea ofperforming an “isothermal” nucleic acid amplification (NASBA, SDA, TMA,LAMP, Q-beta replicase, rolling circle amplification and 3SR arespecifically mentioned) at a predetermined temperature initially,changing the temperature of the reaction, and then allowing thetemperature to return to the predetermined temperature at least onceduring the reaction. More specifically the document suggests causing atemperature oscillation or “wobble” during the amplification reaction,which is said to “improve the overall time to completion andsignal-to-noise [ratio] of the assay”. The idea was exploredexperimentally using the TMA amplification technique to amplifybacterial RNA. The results showed that, whilst the “wobbled” reactionstarted to amplify target sooner than the truly isothermal reaction,there was still a delay of about 13 minutes before the fluorescencesignal rose above the initial background level.

The present invention aims to provide a novel nucleic acid amplificationtechnique having one or more advantages over existing techniques andwhich, in particular, is able to generate quantitative data.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method of performinga non-isothermal nucleic acid amplification reaction, the methodcomprising the steps of:

-   -   (a) mixing a target sequence with one or more complementary        single stranded primers in conditions which permit a        hybridisation event in which the primers hybridise to the        target, which hybridisation event, directly or indirectly, leads        to the formation of a duplex structure comprising two nicking        sites disposed at or near opposite ends of the duplex; and        performing an amplification process by;    -   (b) using a nicking enzyme to cause a nick at each of said        nicking sites in the strands of the duplex;    -   (c) using a polymerase to extend the nicked strands to as to        form newly synthesised nucleic acid, which extension with the        polymerase recreates nicking sites;    -   (d) repeating steps (b) and (c) as desired so as to cause the        production of multiple copies of the newly synthesised nucleic        acid;

characterised in that the temperature at which the method is performedis non-isothermal, and subject to shuttling, a plurality of times,between an upper temperature and a lower temperature during theamplification process of steps (b)-(d), wherein at the uppertemperature, one of said polymerase or nicking enzyme is more activethan the other of said enzymes, such that there is a disparity in theactivity of the enzymes, and at the lower temperature the disparity inthe activity of the enzymes is reduced or reversed.

The nicking enzyme and the polymerase will have certain rates ofcatalytic activity. These will vary with temperature. The respectiverates of activity of the enzymes (in terms of moles of substrate reactedper unit time per mg of enzyme at a given substrate concentration) willusually be different at a particular temperature. Each enzyme will havean optimum temperature at which its rate of activity is maximal.Generally speaking, the further the temperature of a reaction mixture isfrom an enzyme's optimum temperature, the slower the rate of activity ofthe enzyme.

The relative favouring of one enzyme over another (so as to achieve adisparity between the rate of activity of the polymerase andnicking-enzyme) can be obtained by using temperature conditions whichpermit greater activity of one of said enzymes than the other, or byusing temperature conditions which are less favourable for one of theenzymes than the other.

By way of explanation, the disparity in the activity of the enzymes isconsidered to be “reversed” if, at the upper temperature one of theenzymes has a higher activity than the other enzyme, whilst at the lowertemperature the other of said enzymes has a higher activity.

In other embodiments, the disparity in the activity of the enzymes atthe upper and lower temperature is not reversed, but merely reduced.Typically the disparity in activity between the enzymes at one of saidupper or lower temperature is reduced by at least 5% at the lower orupper temperature, as appropriate. More preferably the disparity isreduced by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40% or at least 45%. Most preferablythe disparity is reduced by at least 50%, or at least 75%.

For the avoidance of doubt, “enzyme activity” in this context refers to“specific enzyme activity” (μmol substrate reacted min⁻¹ mg⁻¹ enzyme),measured under the same conditions for the polymerase and the nickingenzyme.

In one preferred embodiment, the method of the first aspect of theinvention comprises the use of a set of temperature conditions whereinat one of said upper and lower temperatures, both the nicking enzyme andthe polymerase are substantially active (i.e. for present purposes,operating at a rate which is at least 50%, or higher, of the rate atwhich the enzyme would operate at its optimum temperature in otherwiseidentical conditions; preferably at 60% or higher, more preferably at65% or higher; and most preferably at 70%, or higher, of its rate ofactivity at its optimum temperature); whilst at the other of said lowerand upper temperatures (as the case may be), at least one of either thenicking enzyme or polymerase is substantially inhibited (i.e. operatingat 49% or less of the rate at which the enzyme would operate at itsoptimum temperature in otherwise identical conditions; preferably lessthan 45%, more preferably less than 40%; and most preferably less than35% of its rate of activity at its optimum temperature). In someembodiments the nicking enzyme is substantially inhibited at one of theupper or lower temperature. In some embodiments the nicking enzyme issubstantially inhibited at the upper temperature.

In some embodiments the polymerase is substantially inhibited at one ofthe upper or lower temperature. In some embodiments the polymerase issubstantially inhibited at the lower temperature; in other embodimentsthe polymerase is substantially inhibited at the upper temperature.

The length of time that the reaction mixture is held constant at theupper temperature, or at the lower temperature, may be referred to asthe “dwell time”, and to distinguish between them, one can refer to the“upper temperature dwell time” and the “lower temperature dwell time”.The upper temperature dwell time and the lower temperature dwell timemay be the same, or may be different. If different, the uppertemperature dwell time may be longer or shorter than the lowertemperature dwell time.

A critical parameter for quantitative analysis is how well the generateddata fit a regression line, known as the coefficient of determination(R²). Data are not considered quantitative if they have a poorcoefficient of determination. For present purposes, data are consideredquantitative if their coefficient of determination is equal to orgreater than 0.850, typically equal to or greater than 0.900, preferablyequal to or greater than 0.950, more preferably equal to or greater than0.975, and most preferably equal to or greater than 0.990. Thecoefficient of determination (R²) may conveniently be calculated usingthe method described by Pfaffl (2001, Nucl. Acids Res. 29 (9) e45).

Accordingly, a method of performing a nucleic acid amplificationreaction and/or analysing a sample by means of such a reaction, isconsidered quantitative if it generates data which are quantitativeaccording to the foregoing definition. Surprisingly, the method of theinvention is able to generate quantitative data.

The amplification reaction of the invention is preferably performed in amanner generally superficially similar to that known as “NEAR” anddisclosed in EP 2,181,196. However importantly, and quite unlike theNEAR technique, the present method is performed non-isothermally andinvolves repeated shuttling between an upper and a lower temperature.

In some embodiments the upper temperature may relatively favour theactivity of the polymerase over that of the nicking enzyme, and thelower temperature will relatively favour the activity of the nickingenzyme over that of the polymerase. Surprisingly however, the inventorshave found that the “temperature preferences” can be fully reversed,such that in some embodiments the upper temperature may relativelyfavour the activity of the nicking enzyme over that of the polymeraseand the lower temperature may relatively favour the activity of thepolymerase over that of the nicking enzyme.

Without being bound by any particular theory, it appears that byappropriate selection of a polymerase and a nicking enzyme withdifferent temperature optima it is possible to have the uppertemperature of the amplification reaction relatively favour either thepolymerase or the nicking enzyme, and vice versa in relation to thelower temperature of the amplification reaction.

Without being bound by any particular theory, it is further hypothesisedby the inventors that a possible mechanism for the rapid amplificationachieved by the method of the present invention invokes causing areduction in the activity of one or other of the nicking enzyme or thepolymerase, by using a temperature which is considerably sub-optimal forthe enzyme, leading to an accumulation of potential substrate molecules.When the temperature of the reaction mixture is adjusted to atemperature which is closer to optimal for the enzyme in question, theactivity of the enzyme is significantly enhanced which, in conjunctionwith the relatively high concentration of accumulated substrate, resultsin a greatly accelerated rate of reaction. In simplistic terms, theaverage rate of reaction of this “quick/slow” format is greater than theaverage rate of reaction achievable using a “steady state” system with aconstant, or relatively slowly-changing, temperature.

It will be apparent to the person skilled in the art that it may bedesirable that the optimum temperature of the nicking enzyme bedifferent (higher or lower) from that of the polymerase used in themethod of the invention.

Typically the respective optimum temperatures of the nicking enzyme andthe polymerase should differ by at least 1° C., preferably by at least3° C., more preferably by at least 5° C., and most preferably by atleast 10° C. Conveniently the respective optimum temperatures willdiffer by an amount in the range 10-30° C., more typically in the range10-25° C.

There is no absolute requirement that the optimum temperature of thepolymerase is higher than that of the nicking enzyme. Thus, for example,there are embodiments of the invention in which the reaction utilises apolymerase (e.g. obtained from a psychrophilic source) which has a loweroptimum temperature than that of the nicking enzyme, whilst in otherembodiments the polymerase has a higher optimum temperature than that ofthe nicking enzyme.

Thus, in general, the upper temperature is preferably selected so as torelatively favour a sequence-specific polymerase-mediated extensionphase (i.e. formation of a complex between the polymerase and thehybridised initial primer/target duplex, followed by thepolymerase-mediated extension of the primer; and almost immediatelythereafter, extension of the opposite primer hybridised to the extendedinitial primer). The use of an elevated temperature tends to reduceprimer dimer formation and aberrant amplification of undesiredmis-hybridised duplexes. The polymerase is conveniently selected so asto be sufficiently stable at the upper temperature as to perform theprimer extension throughout the duration of the reaction withoutsignificant diminution of activity. For present purposes, “significantdiminution” means a decline of 50% or more in specific enzyme activityof the polymerase.

The lower temperature is preferably selected so as to permit the nickingenzyme to cut the nick sites on the duplex. The nicking enzyme typically(but not necessarily) has an optimum temperature which is lower thanthat of the polymerase, hence the transition to the lower temperaturetypically moves the reaction temperature closer to the optimumtemperature of the nicking enzyme.

In some embodiments of the method of the invention as exemplifiedherein, the upper temperature is preferably in the range 50.0-64.0° C.,more preferably in the range 55.0-63.0° C. However, those skilled in theart will appreciate that the preferred “upper temperature” may varydepending on the identity of the enzymes present and possibly also onthe length and sequence of the primers and/or the intended amplificationtarget.

For example, in some embodiments, the upper temperature could be as highas 68° C. but, in those conditions:

-   -   (a) one would normally wish to use a thermal shuttling profile        with a reduced dwell time at the upper temperature (e.g. no more        than about 1-2 seconds per shuttle); and    -   (b) such a high upper temperature works well only with        relatively high copy number of target sequence in the sample        (e.g. about 10³ copies or higher).

In the method of the invention as exemplified herein, the lowertemperature is preferably in the range 20.0-58.5° C., more preferably inthe range 35.0-57.9° C. Again, however, as noted above, those skilled inthe art will appreciate that the preferred “lower temperature” may varydepending on the identity of the enzymes present, and possibly also thelength and sequence of the primers and/or the intended amplificationtarget.

As a general rule, as is well known to those skilled in the art, thestringency of hybridisation increases with increasing temperature(within limits), such that higher temperatures will generally reducenon-specific interactions such as between mis-matched primers andnon-complementary polynucleotide sequences present in the sample. Thus ahigher temperature for hybridisation reactions will normally bepreferable to a lower temperature, as long as the temperature does notexceed the melting temperature of the specific primer/target sequencehybridisation.

Desirably, in preferred embodiments the difference in temperaturebetween the upper temperature and the lower temperature will be in therange 4-12° C., more preferably in the range 4-10° C., and mostpreferably in the range 4-8° C.

Generally, although not necessarily, it may be preferred for thereaction mixture to be held at the upper temperature for a shorterperiod of time (the “dwell time”) than that for the lower temperature,although the “dwell time” at the upper and lower temperatures could beequal or even, in other embodiments, the dwell time at the uppertemperature might be longer than that for the lower temperature—althoughthis is generally not preferred.

It is envisaged that, within certain limits, in general the higher thefrequency of the thermal shuttling, the faster the amplificationreaction will proceed. Thus the duration of one complete thermal shuttlewill preferably be less than 3.0 minutes, more preferably less than 2.0minutes, and most preferably less than 1.0 minute. Most advantageously,the duration of a thermal shuttle will be less than 45 seconds and mostpreferably less than 30 seconds. A minimum duration of a thermal shuttlewill typically be at least 1 second, preferably at least 2 seconds, andmore preferably at least 5 seconds. A typical preferred duration for onecomplete thermal shuttle will be between 5 and 30 seconds, preferablybetween 5 and 20 seconds, and most preferably between 5 and 15 seconds.

A typical preferred dwell time at the upper temperature might be between1 and 10 seconds, preferably 1-5 seconds, and most preferably 1-3seconds.

A typical preferred dwell time at the lower temperature might be between2 and 40 seconds, more preferably between 3 and 30 seconds, and mostpreferably between 3 and 15 seconds.

The time taken to shuttle between the upper and lower temperatures ispreferably kept substantially to a minimum. It is envisaged that thetypical volume of an amplification reaction mixture will be less than500 μl, probably less than 250 μl and, given that the upper and lowertemperatures will typically be less than 10° C. apart, it should bepossible and preferred to transition from the lower to upper temperature(or vice versa) in about 0.5-10.0 seconds, more preferably in the range1-5 seconds.

Conveniently the duration/temperature profile of each of the pluralityof shuttles is essentially identical—this simplifies performance of themethod. Thus, for example, each of the plurality of thermal shuttleswill conveniently have the same overall duration, the same dwell time atthe upper temperature, the same dwell time at the lower temperature,etc.

However, in some embodiments (especially those in which there isreal-time detection of the direct or indirect product's of theamplification reaction), it may be desirable to alter the profile of thethermal shuttling during the course of the reaction, so that not all ofthe shuffles are identical. More specifically, if real-timequantification of the amplification reaction product/s (whether director indirect) indicates that the reaction is proceeding more slowly thanis desirable, this information might be fed back to the thermalregulation apparatus which regulates the temperature of the reactionmixture, causing the apparatus to adjust the profile of the thermalshuttling, so as to increase the rate of reaction. This might berequired if, for example, the target sequence is present in very lowcopy number. The apparatus might adjust the thermal shuttling profile byincreasing or decreasing the upper and/or lower temperature, and/orincreasing or decreasing the dwell time at the upper and/or lowertemperature. It is also feasible that the apparatus might increase ordecrease the time taken to transition between the upper and lowertemperatures (i.e. increase or decrease the time of either the upwardtemperature transition, or the downward temperature transition, orboth).

The thermal shuttling may be commenced substantially immediately afterall the necessary components of the reaction mixture have been broughttogether.

Alternatively, the thermal shuttling may be commenced after a delayinterval. For example, it is possible, and potentially desirable, thatthe reaction mixture might be held at an elevated temperature (whichmight be the same as, or different to, the upper temperature used in thethermal shuttling). As an illustration, such a delay interval might befrom e.g. 5 seconds to 1 or 2 minutes.

Further, the thermal shuttling may conveniently be performedsubstantially continuously during the amplification reaction, or may besubject to one or more pauses. Typically, and preferably, once commencedthe thermal shuttling will not be interrupted until the amplificationreaction has reached a desired time point, typically by when adetectable fluorescence (or other) signal has been obtained and whichallows advantageously quantitative determination of the amount and/orconcentration of the target sequence in the sample.

The thermal shuttling of the amplification reaction mixture mayconveniently be effected using automated thermal regulation apparatus,such as is commercially available for performing thermal cycling in PCR.Clearly the temperature profiles generated by the apparatus will needmatching to the preferred conditions applicable in performance of thepresent invention.

In a second aspect, the invention provides a reaction mixture forperforming a nucleic acid amplification, the mixture comprising a targetsequence to be amplified, two or more primers, one of said primers beingcomplementary to a first strand of the target and the other of saidprimers being complementary to a second strand of the target, a DNApolymerase, and a nicking enzyme; said reaction mixture being in thermalregulation association with programmable temperature regulation means,said temperature regulation means being programmed to perform thermalshuttling between an upper and lower temperature, as defined previouslyin relation to the first aspect of the invention.

In a third aspect, the invention provides a method of determining theamount and/or concentration of a target polynucleotide in a sample, themethod comprising the steps of: performing an amplification reaction inaccordance with the first aspect of the invention defined above toamplify the target and detecting, in a quantitative manner, the director indirect product/s of the amplification reaction, so as to allow adetermination of the amount and/or concentration of the targetpolynucleotide in the sample.

The amplification process of the method of the invention may be appliedto generally known and conventional amplification techniques includingSDA and NEAR, which utilise a polymerase and a nicking enzyme.

Thus, for example, the amplification process may be based on theamplification process employed in strand displacement amplification, orbased on that used in NEAR or indeed any other nucleic acidamplification process which relies on the creation of a single strandednick and subsequent extension from the 3′ end of the nicked strand.Other than the teachings of the prior art in relation to maintenance ofconstant temperature during the amplification, the teachings of theprior art in relation to the amplification stages of SDA or NEAR will,in general, be equally applicable to the amplification process of themethod of the present invention.

The method of the present invention is an improvement of theamplification technique named Selective Temperature AmplificationReaction (or “STAR”) described in WO2018/002649. The method of thepresent invention, in preferred embodiments, permits real-timequantitative detection of target sequences, and is referred to herein as“qSTAR”, although this is not intended to indicate that the method ofthe invention will provide quantitative real-time results under allconditions.

Preferably step (a) comprises mixing a sample containing double strandedtarget with two single stranded primers, one of said primers beingcomplementary to a first strand of the target, and the other of saidprimers being complementary to a second strand of the target, such thatthe two primers hybridise to the target and the free 3′ ends of saidprimers face towards one another.

The two primers may conveniently be described as ‘forward’ and ‘reverse’primers.

Desirably both the forward and reverse primers will comprise thesequence of a nicking enzyme recognition site. Typically the nickcreated by a nicking enzyme will be just outside and typically 3′ of thenicking enzyme recognition site.

In a preferred embodiment, the forward primer will comprise a portion ator near its 3′ end which is complementary to, and can hybridise with,the 3′ end of the target sequence antisense strand, whilst the reverseprimer comprises a portion at or near its 3′ end which is complementaryto, and can hybridise with, the 3′ end of the target sequence sensestrand.

In this way, a nicking enzyme recognition site is introduced at oppositeends of the target sequence, and amplification of the target sequence(together with any intervening sequence of the primers downstream of thenick site) is accomplished by performing multiple cycles of polymeraseextension of the forward and reverse primers so as to form a doublestranded nicking enzyme recognition site, and by nicking of the siteswith a nicking enzyme, allowing further extension of the nicked primersby a polymerase etc., essentially as disclosed in, for example, US2009/0017453, the content of which is herein incorporated by reference.

The target may be single stranded, double stranded, or comprise amixture of the two. The target may comprise RNA, DNA or a mixture of thetwo. In particular the target might incorporate one or more modifiednucleotide triphosphates (i.e. a nucleotide triphosphate not normallyfound in naturally occurring nucleic acids), although this is notessential and indeed not preferred.

The target may be selected from the following non-exhaustive list:genomic nucleic acid (which term encompasses the genomic nucleic acid ofany animal, plant, fungus, bacterium or virus), plasmid DNA,mitochondrial DNA, cDNA, mRNA, rRNA, tRNA, or a syntheticoligonucleotide or other nucleic acid molecule.

In particular, the method may additionally comprise an initial reversetranscription step. For example, RNA (e.g. viral genomic RNA, orcellular mRNA, or RNA from some other source) may be used to synthesiseDNA or cDNA using a reverse transcriptase by methods well-known to thoseskilled in the art. The DNA may then be used as a target sequence in themethod of the invention. The original RNA will typically be degraded bythe ribonuclease activity of reverse transcriptase, but if desiredadditional RNase H may be added after reverse transcription has beencompleted. RNA molecules are often present in samples at greater copynumber than corresponding (e.g. genomic) DNA sequences, hence it may beconvenient to make DNA transcripts from the RNA molecule in order toeffectively increase the copy number of the DNA sequence.

The “target sequence” is the sequence of bases in the target nucleicacid, and may refer to the sense and/or antisense strand of a doublestranded target, and also encompasses, unless the context dictatesotherwise, the same base sequence as reproduced or replicated inamplified copies, extension products or amplification products of theinitial target nucleic acid.

The target sequence may be present in any kind of sample e.g. biologicalor environmental (water, air etc.). A biological sample may be, forexample, a food sample or a clinical sample. Clinical samples mayinclude the following: urine, saliva, blood, serum, plasma, mucus,sputum, lachrymal fluid or faeces.

The sample may or may not be subject to processing before beingcontacted with the primers. Such processing may include one or more of:filtration, concentration, partial-purification, sonication, chemicallysis and the like. Such processes are well-known to those skilled inthe art.

The method of the present invention involves the use of a nick site andmeans for creating a nick at the nick site. A “nick” is the cleavage ofthe phosphodiester backbone of just one strand of a fully, or at leastpartially, double stranded nucleic acid molecule. The nick site is thelocation in the molecule where a nick is made.

In preferred embodiments a “nicking recognition site” will be presentat, within, or next to a nick site. (“Next to” in this context meansthat the nearest base of the nicking recognition site is within 10 basesof the nick site, preferably within 5 bases of the nick site).

The nicking recognition site may comprise at least one strand of therecognition site of a restriction endonuclease, and the nick site maycomprise at least one strand of a nucleic acid base sequence which, whenpresent as a double stranded molecule, is cut by a restrictionendonuclease. Typically a restriction endonuclease will cut both strandsof a double stranded nucleic acid molecule. In the present invention, adouble stranded break can be avoided by the incorporation of one or moremodified bases at or near to the nick site, which modified bases rendera strand of nucleic acid not susceptible to cleavage by the restrictionendonuclease. In this way a restriction endonuclease, which usually cutsboth strands of a double stranded nucleic acid molecule, can be used tointroduce a single stranded nick into a double stranded molecule.Modified bases and the like suitable for achieving this are well-knownto those skilled in the art and include, for example, all alphaphosphate modified nucleoside triphosphates and alpha borano modifiednucleoside triphosphates, specifically; 2′-deoxyadenosine5′-O-(thiotriphosphate), 5′-triphosphate, 2′-deoxyuridine5′triphosphate, 7-deaza-2′deoxyguanosine 5′-triphosphate,2′deoxyguanosine-5′-O-(1-boranotriphosphate) and others. Triphosphatesincluding the modified base may be present within a reaction mixtureused to perform the amplification process, so that modified bases areincorporated at relevant positions during subsequent rounds ofamplification to prevent the formation of a double-stranded sitecleavable by the endonuclease.

In preferred embodiments however the nick is made at the nick site bymeans of a nicking enzyme. Nicking enzymes are molecules which, undernormal circumstances, make only a single stranded break in a doublestranded nucleic acid molecule. The nicking enzyme typically has anicking recognition site and the nick site may be within the nickingrecognition site or may be either 5′ or 3′ of the recognition site. Manynicking enzymes are known to those skilled in the art and arecommercially available. A non-exhaustive list of examples of nickingenzymes includes: Nb.Bsml, Nb.Bts, Nt.Alwl, Nt.BbvC, Nt.BstNBI, andNt.Bpu101. The latter enzyme is commercially available from ThermoFisherScientific; the others are available from e.g. New England Biolabs.

In preferred embodiments, the nicking enzyme is introduced into thereaction mixture at the outset of the method (e.g. within one minute ofcontacting the sample with primers and DNA polymerase). However, in someinstances it may be desirable to introduce the nicking enzyme into thereaction mixture after a longer delay (e.g. to allow the temperature tofall closer to the optimum temperature of the nicking enzyme).

The method of the invention involves the use of a DNA polymerase.Preferably, but not necessarily, the method of the invention maycomprise the use of at least one thermophilic DNA polymerase (i.e.having an optimum temperature in excess of 60° C.). Preferably the DNApolymerase is a strand displacing polymerase. Preferably the DNApolymerase has no exonuclease activity. Preferably the DNA polymerase isa strand-displacing, polymerase with no exonuclease activity, and isalso preferably thermophilic.

Examples of preferred DNA polymerases include Bst polymerase, VENT® DNApolymerase, 9° N polymerase, “MANTA″Tm 1.0 polymerase (Qiagen), BstXpolymerase (Qiagen), SD polymerase (Bioron GmbH), Bsm DNA polymerase,large fragment (ThermoFisher Scientific), Bsu DNA polymerase, largefragment (NEB), and “ISOPOL”™” polymerase (from ArcticZymes).

Table 1 below gives examples of combinations of a nicking enzyme and aDNA polymerase, together with the suggested upper and lower temperatureto use in performing the method of the invention using the exemplifiedenzyme combinations. Although the table lists specific DNA polymerases,these are by way of example only and any strand displacing exonucleaseminus, polymerase with activity in the stated temperature range wouldsuffice such as: Deep Vent (exo-), Bst DNA Polymerase I, II, and III,“MANTA”™ 1.0 DNA Polymerase, Bst X DNA Polymerase, Bsm DNA Polymerase,“ISOPOL”™ DNA Polymerase.

TABLE 1 Suggested Suggested Suggested Upper Lower Nicking SuggestedTemperature Temperature Enzyme(s) DNA Polymerase(s) (° C.) (° C.)Nt.BstNBI Bst DNA Polymerase, 62° C. 57° C. Large Fragment Nt.BstNBI BsuDNA 45° C. 38° C. Polymerase, Large Fragment Nt.Alwl Bst DNA Polymerase,62° C. 57° C. Large Fragment Nt.Alwl Bsu DNA Polymerase, 45° C. 38° C.Large Fragment Nt. BsmAl Bst DNA Polymerase, 60° C. 55° C. LargeFragment Nt. BsmAl Bsu DNA Polymerase, 44° C. 37° C. Large FragmentNt.BspQ1 Bst DNA Polymerase, 62° C. 52° C. Large Fragment Nt. BspQ1 BsuDNA 44° C. 37° C. Polymerase, Large Fragment

In some embodiments, the method of the invention may convenientlycomprise a pre-amplification or enrichment step. This is a step in whichthe target sequence is contacted with forward and reverse primers andDNA polymerase, but no nicking enzyme. This typically lasts for about1-5 minutes and produces an initial (linear) amplification of the targetsequence of about 1,000 fold, which can be especially useful if thetarget sequence is present in the sample at low copy number.

In some embodiments, the pre-amplification or enrichment step isperformed using a mesophilic DNA polymerase such as Exo-Minus Klenow DNAPolymerase or Exo-Minus psychrophile DNA polymerase from Cenarchaeumsymbiosum, at a temperature below 50° C., and the mixture issubsequently heated above 50° C. to denature or inactivate thethermolabile DNA polymerase, and then a thermophilic DNA polymerase isadded for downstream amplification.

Typically, the method of the invention comprises a detection step, inwhich one or more of the direct or indirect products of theamplification process is detected and optionally quantified, thisindicating the presence and/or amount of the target in the sample. Thereare a great many suitable detection and/or quantification techniquesknown, including: gel electrophoresis, mass spectrometry, lateral flowcapture, incorporation of labelled nucleotides, intercalating or otherfluorescent dyes, enzyme labels, electrochemical detection techniques,molecular beacons and other probes, especially specifically hybridisingoligonucleotides or other nucleic acid containing molecules.

The product or products which are detected in the detection step may bereferred to herein as the “detection target”. The ‘target’ in relationto the detection step, is not necessarily the same as the ‘target’ inthe amplification process and indeed the two molecules will usually bedifferent to at least some extent, although they may have some sequence(typically 10-20 bases) in common, where the detection target comprisesa nucleic acid molecule or oligonucleotide.

Nucleic acid detection methods may employ the use of dyes that allow forthe specific detection of double-stranded DNA. Intercalating dyes thatexhibit enhance fluorescence upon binding to DNA or RNA are well known.Dyes may be, for example, DNA or RNA intercalating fluorophores and mayinclude inter alia the following: acridine orange, ethidium bromide,Pico Green, propidium iodide, SYBR® I, SYBR® II, SYBR® Gold, TOTO-3 (athiaxole orange dimer) OLIGREEN™ and YOYO™ (an oxazole yellow dimer).

Nucleic acid detection methods may also employ the use of labellednucleotides incorporated directly into the detection target sequence orinto probes containing sequences complementary or substantiallycomplementary to the detection target of interest. Suitable labels maybe radioactive and/or fluorescent and can be resolved in any of themanners conventional in the art. Labelled nucleotides, which can bedetected but otherwise function as native nucleotides (e.g. arerecognised by and may act as substrates for, natural enzymes), are to bedistinguished from modified nucleotides, which do not function as nativenucleotides.

The presence and/or amount of target nucleic acids and nucleic acidsequences may be detected and monitored using molecular beacons.Molecular beacons are hair-pin shaped oligonucleotides containing afluorophore at one end and a quenching dye (“quencher”) at the oppositeend. The loop of the hair-pin contains a probe sequence that iscomplementary or substantially complementary to a detection targetsequence and the stem is formed by the annealing of self-complementaryor substantially self-complementary sequences located either side of theprobe sequence.

The fluorophore and the quencher are bound at opposite ends of thebeacon. Under conditions that prevent the molecular beacon fromhybridizing to its target or when the molecular beacon is free insolution, the fluorophore and quencher are proximal to one another,preventing fluorescence. When the molecular beacon encounters adetection target molecule, hybridization occurs; the loop structure isconverted to a stable, more rigid conformation causing separation of thefluorophore and quencher allowing fluorescence to occur (Tyagi et al.1996, Nature Biotechnology 14: 303-308). Due to the specificity of theprobe, the generation of fluorescence is substantially exclusively dueto the presence of the intended amplified product/detection target.

As a general rule, molecular beacons work better at lower hybridisationtemperatures, as the signal to noise ratio decreases with increasingtemperature. This is because at lower temperatures theself-complementary “stem” parts of the molecular beacon remain firmlyhybridised, allowing the quencher to quench the fluorophore, but as thetemperature increases the stem parts of the molecule can start to melt,allowing non-specific fluorescence background “noise” to increase.

Molecular beacons are highly specific and can distinguish nucleic acidsequences differing by a single base (e.g. single nucleotidepolymorphisms). Molecular beacons can be synthesized with differentcoloured fluorophores and different detection target complementarysequences, enabling several different detection targets in the samereaction to be detected and/or quantified simultaneously, allowing“multiplexing” of a single PoC assay to detect a plurality of differentpathogens or biochemical markers. For quantitative amplificationprocesses, molecular beacons can specifically bind to the amplifieddetection target following amplification, and because non-hybridizedmolecular beacons do not fluoresce, it is not necessary to isolateprobe-target hybrids to quantitatively determine the amount of amplifiedproduct. The resulting signal is proportional to the amount of theamplified product. This can be done in real time. As with other realtime formats, the specific reaction conditions must be optimized foreach primer/probe set to ensure accuracy and precision.

The production or presence of detection target nucleic acids and nucleicacid sequences may also be detected and monitored by fluorescenceresonance energy transfer (FRET). FRET is an energy transfer mechanismbetween two fluorophores: a donor and an acceptor molecule. Briefly, adonor fluorophore molecule is excited at a specific excitationwavelength. The subsequent emission from the donor molecule as itreturns to its ground state may transfer excitation energy to theacceptor molecule (through a long range dipole-dipole interaction). FRETis a useful tool to quantify molecular dynamics, for example, in DNA-DNAinteractions as seen with molecular beacons. For monitoring theproduction of a specific product a probe can be labelled with a donormolecule on one end and an acceptor molecule on the other.Probe-detection target hybridization brings a change in the distance ororientation of the donor and acceptor and a change in the FRETproperties is observed. (Joseph R. Lakowicz. “Principles of FluorescentSpectroscopy”, Plenum Publishing Corporation, 2^(nd) edition (Jul. 1,1999)).

The production or presence of detection target nucleic acids may also bedetected and monitored by lateral flow devices. Lateral Flow devices arewell known. These devices generally include a solid phase fluidpermeable flow path through which fluid flows by capillary force.Examples include, but are not limited to, dipstick assays and thin layerchromatographic plates with various appropriate coatings. Immobilized inor on the flow path are various binding reagents for the sample, bindingpartners or conjugates involving binding partners for the sample, andsignal producing systems. Detection of analytes can be achieved inseveral different ways including: enzymatic detection, electrochemicaldetection, nano-particle detection, colorimetric detection, andfluorescence detection. Enzymatic detection may involve enzyme-labelledprobes that are hybridized to complementary or substantiallycomplementary nucleic acid detection targets on the surface of thelateral flow device. The resulting complex can be treated withappropriate markers to develop a readable signal. Nanoparticle detectioninvolves bead technology that may use colloidal gold, latex andparamagnetic nanoparticles. In one example, beads may be conjugated toan anti-biotin antibody. Target sequences may be directly biotinylated,or target sequences may be hybridized to a sequence specificbiotinylated probes. Gold and latex give rise to colorimetric signalsvisible to the naked eye and paramagnetic particles give rise to anon-visual signal when excited in a magnetic field and can beinterpreted by a specialized reader.

Fluorescence-based lateral flow detection methods are also known, forexample, dual fluorescein and biotin-labelled oligo probe methods, orthe use of quantum dots.

Nucleic acids can also be captured on lateral flow devices. Means ofcapture may include antibody dependent and antibody independent methods.Antibody-independent capture generally uses non-covalent interactionsbetween two binding partners, for example, the high affinity andirreversible linkage between a biotinylated probe and a streptavidincapture molecule. Capture probes may be immobilized directly on lateralflow membranes.

The entire method of the invention, or at least the amplificationprocess portion of the method, may be performed in a reaction vessel(such as a conventional laboratory plastics reagent tube e.g. fromEppendorf®) or may be performed in and/or on a solid support. The solidsupport may be porous or non-porous. In a particular embodiment thesolid support may comprise a porous membrane material (such asnitrocellulose or the like). More especially the solid support maycomprise or form part of a porous lateral flow assay device, asdescribed above. Alternatively, the solid support may comprise or formpart of a microfluidics-type assay, in which one or more solidnarrow-bore capillary tubes are used to transport a liquid along anassay device.

In preferred embodiments, all or at least part of the method of theinvention may be performed using a point-of-care (PoC) assay device. APoC device typically has the following characteristics: it is cheap tomanufacture, is disposed of after a single use, is generallyself-contained not requiring any other apparatus or equipment to performor interpret the assay and, desirably, requires no clinical knowledge ortraining to use.

The method of the invention especially lends itself to performance usinga PoC-type device since, in typical embodiments, the difference intemperature between the upper and lower temperatures of the thermalshuttle is quite small. As a result, relatively simple thermalshuttling/temperature regulation is sufficient, in contrast say, toperforming qPCR.

Nevertheless, the amplification method of the present invention couldalso be used in a lab-based system, rather than a PoC device, in placeof qPCR and can typically achieve quantitative results much more quicklythan can be achieved by performing qPCR.

Examples of primers suitable for use in the invention are disclosedherein. Other examples which may be suitable for use in the method ofthe invention are disclosed in, inter alia, US 2009/0017453,US2013/0330777, and EP 2,181,196, the content of which is incorporatedherein by reference. The person skilled in the art will be readily ableto design other primers suitable for the amplification of other targetsequences without undue experimentation.

As explained elsewhere, primers of use in the invention will preferablycomprise not only a target complementary portion, but also a nickingendonuclease binding site and nicking site, and a stabilizing portion.

Primers of use in the method of the invention may comprise modifiednucleotides (i.e. nucleotides not found in naturally occurring nucleicacid molecules). Such modified nucleotides may conveniently be presentin the target complementary portion of the primer, and/or elsewhere inthe primer. Preferred examples of modified nucleotides are 2′-modifiednucleotides, especially 2′-O-methyl modified nucleotides, although manyother modified nucleotides are known to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the invention will now be described by way ofillustrative example and with reference to the accompanying drawings, inwhich:

FIGS. 1A and 1B are schematic representations of the initiation phaseand exponential amplification phase respectively of a nucleic acidamplification reaction suitable for performing the method of theinvention;

FIG. 2 is a graph (temperature in 0° against time, in minutes)illustrating a typical temperature profile for a reaction mixture duringperformance of the method of the Invention;

FIG. 3 is a schematic representation of a typical embodiment of a primeroligonucleotide molecule useful in performing the method of theinvention;

FIGS. 4, 5A, 5B and 5C are graphs of (background subtracted)fluorescence (in arbitrary units) against time (seconds) foramplification reactions performed in accordance with the method of theinvention, using primer molecules comprising no 2′-O-methylated bases(FIG. 4 ), or primer molecules comprising one, six, or seven2′-O-methylated bases (FIGS. 5A, 5B and 5C respectively);

FIG. 6 is a scatter plot showing the average time to achieveamplification (A-r, in minutes), as judged by generation of afluorescence signal above a background threshold, for a method inaccordance with the invention (left hand plot), and a method performedin accordance with the STAR protocol disclosed in WO2018/002649 (middleplot), or an isothermal reaction protocol (right hand plot);

FIGS. 7 and 8 are schematic representations of, respectively, apolymerase activity assay and a nicking activity assay, of use incharacterising the method of the invention;

FIGS. 9A and 9B are graphs of average (of three replicates) of relativefluorescence (in arbitrary units) against time (in minutes) of apolymerase activity assay (FIG. 9A) or a nicking activity assay (FIG.9B), conducted at a variety of temperatures;

FIGS. 10A-11D are graphs of relative fluorescence (arbitrary units)against cycle number for amplification reactions attempted using variousdifferent polymerase enzymes using conventional PCR conditions (FIGS.10A, 10B) or “gSTAR” thermal shuttling conditions in accordance with theinvention but in the absence of a nicking enzyme (FIGS. 11A-11D);

FIGS. 12, 13A and 13B show the data obtained from performingconventional gPCR amplification (FIG. 12 ), or “gSTAR” amplification inaccordance with the method of the invention (FIG. 13A), using startingsamples of unknown concentration, with a summary of the results in FIG.136 ;

FIGS. 14A-14D are graphs of fluorescence (arbitrary units) against time(seconds), showing the results of amplification reactions performedaccording to the method of the invention over different temperatureranges;

FIG. 15 is a graph of fluorescence (arbitrary units) against time(minutes), showing the results of amplification reactions performedaccording to the method of the invention at temperatures in the range38-45° C.; and

FIG. 16 is a graph of fluorescence (arbitrary units) against time(minutes), showing the results of amplification reactions performedaccording to the method of the invention, using a reverse transcribedRNA target sequence.

EXAMPLES Example 1: Protocol for Testing Quantitative SelectiveTemperature Amplification Reaction (gSTAR)

Quantifying gene expression by Selective Temperature AmplificationReaction (STAR) as described in WO2018/002649, or other similarlyrelated DNA/RNA amplification technologies such as PCR, SDA, or anisothermal amplification technique, would be, at best, unreliable. Theamount of product produced would reach a plateau that is not directlycorrelated with the amount of target DNA in the initial starting sample.By establishing a zonal effect of controlled temperature shuttling on anamplification reaction, quantitative amplifications can be achieved witha strand displacement polymerase and nicking endonuclease in which theamplified product is directly related to the initial starting amount ofDNA, RNA, or other known nucleic acids. A nicking enzyme-based selectivetemperature amplification reaction, in accordance with the invention, isreferred to herein as quantitative Selective Temperature AmplificationReaction (gSTAR). The protocol is further described below unlessotherwise noted.

Enzymes, Oligonucleotides, and Target

Chlamydia trachomatis (Ct) was used as the initial target for thedevelopment of the gSTAR mechanism. Chlamydia trachomatis Serovar J(ATCC VR-886) genomic DNA was acquired from American Type CultureCollection (Manassas, VA). The open reading frame 6 region of thecryptic plasmid was amplified with primers gSTARctF61a (SEQ ID NO: 15′-CGACTCCATATGGAGTCGATTTCCCCGAATTA-3′) and gSTARctR61c (SEQ ID NO: 25′-GGACTCCACACGGAGTCTTTTTCCTTGTTTAC-3′). The resulting DNA template wasdetected using a molecular beacon qSTARctMB1 (SEQ ID NO: 3,5′-FAM/ccattCCTTGTTTACTCGTATTTTTAGGaatgg/BHQ1-3′) as described in EP No.0728218. Bst X DNA polymerase was purchased from Qiagen (Beverly, MA).Nt.BstNBI nicking endonuclease was purchased from New England BioLabs(Ipswich, MA) and is described in U.S. Pat. No. 6,191,267. The samepolymerase and nicking endonuclease were also used in the other examplesdescribed herein, unless otherwise stated.

Oligonucleotides and molecular beacons were synthesized by IntegratedDNA Technologies (Coralville, IA) and Bio-Synthesis (Lewisville, TX).The general features of the primers used in the gSTAR reactions are asdescribed in WO2018/002649.

A summary of the oligonucleotides and amplification mechanism found in areaction in one embodiment of the present invention comprises (i) atarget nucleic acid molecule; (ii) two or more primer oligonucleotidemolecules comprising some number of oligonucleotides that arecomplementary to the target nucleic acid molecule and (iii) a sitewithin the primer that can be nicked by a nicking enzyme. The methodinvolves contacting a target nucleic acid molecule with a polymerase,two or more primer oligonucleotides, each of which specifically binds toa complementary sequence on the target nucleotide molecule, and anicking enzyme, under selective temperature amplification conditions,generating a detectable amplicon that comprises at least a portion ofthe target sequences that a primer oligonucleotide had bound to. Theoverall qSTAR reaction can be understood to undergo two distinct phases;initiation and exponential amplification, illustrated schematically inFIGS. 1A and 1B respectively. The initiation phase is the initialformation of a protein-primer duplex from which initial extension forexponential amplification can occur. The exponential phase is when thenicking enzyme becomes active along with the polymerase leading toexponential amplification. In FIG. 1A, initial contact of the primer toa target nucleic acid occurs (step a), followed by polymerase extension(step b) which generates the forward initiation template (c). Theopposite strand primer binds (step d) to the newly generated forwardinitiation template, extending (step e) in the direction toward, andthrough, the initiation template's nick site. This initiation can occursimultaneously on both the forward and reverse strands. This initialprocess can be understood as predominantly involving the polymerase forextension, but with essentially little or no involvement of the nickingenzyme.

In FIG. 1A, the target is shown as being single stranded. This is forthe purposes of clarity and simplicity. In reality, the method of theinvention is performed without requiring the use of high temperatures to‘melt’ or separate the strands of double stranded targetpolynucleotides—rather, primers are able to associate with the (doublestranded) target molecule by taking advantage of localised relaxation ofthe hydrogen bonding between the strands—a phenomenon known as“breathing”.

At a (in this embodiment, lower) second selective temperature, nickingis favoured on either strand allowing the strand displacing polymeraseto extend toward the opposite primer and through the nick site. Thiscycle of nicking/polymerase extension results in the formation of theExponential Duplex (FIG. 1B). This Exponential Duplex then feeds into abidirectional amplification as each new template generated from a nickand extension becomes a target for another primer. The temperature isshuttled back to the initiation phase for polymerase specific extension,limiting background amplification and controlling exponentialamplification in discreet phases.

By controlling the temperatures, and thus the activity of the polymeraseand nicking endonuclease, the applicants have achieved a method forrapid and controlled amplification, allowing for quantitation of unknowntarget input.

FIG. 2 shows a typical temperature profile (° C. against time, inminutes) for one embodiment of a amplification reaction in accordancewith the invention. In the illustrated embodiment, the polymerase has ahigher optimum temperature than that of the nicking enzyme. The uppertemperature is 63° C., the lower temperature is 57° C. The dwell time atthe lower temperature (about 5 seconds) is longer than the dwell time(about 2 seconds) at the upper temperature. Each complete temperatureshuttle lasts about 8-9 seconds, such that approximately 7 thermalshuttles are completed per minute. In the upper temperature half of theshuttle (>60° C.) the initiation phase of the reaction (see FIG. 1B) isfavoured and predominates. Those skilled in the art will appreciate thatthere is no sharp temperature distinction between the two phases of theamplification reaction, and the dividing line illustrated in FIG. 2 issimply to aid understanding.

The approach of quantitative selective temperature amplification hassurprisingly resulted in a quantitative, rapid, specific, and high yieldamplification reaction with significantly greater performance thanpreviously existing methods, as will be further explained andillustrated in greater detail below.

Amplification Conditions

The basic qSTAR mixture contained two primers, polymerase, and nickingenzyme (referenced above). The reactions were performed in a finalvolume of 25 μl, including 1.0 μM of the forward primer, 0.5 μM of thereverse primer, 0.25 μM molecular beacon, 10 μl qSTAR Master Mix and 5μl DNA sample. gSTAR master mix contained the following reagents; 12.5mM MgSO₄, 90 mM Tris-HCl (pH 8.5), 300 μM each dNTPs, 20 mM NH₄OAc, 30mM NaOAc, 2 mM DTT, 0.02% Triton X-100, 15U nicking endonuclease and 60Upolymerase. The temperature of the reactions was controlled between twodiscreet temperature phases to take advantage of inherent enzymeactivities. The initiation phase, consisting primarily of polymeraseactivity, was at the elevated temperature of 62° C. for two seconds. (Atthis temperature the nicking enzyme was largely inhibited—see FIG. 9B).The exponential phase, in which both the polymerase and nicking enzymeare moderately or highly active, was held at 57° C. for five seconds.The total time for a complete shuttle was 15 seconds. This is more thandouble the dwell time at each temperature due to the limits of theapparatus in changing temperature (a more responsive instrument wouldallow for faster shuttling between upper and lower temperature).Amplification and qSTAR product detection were performed using theAgilent Mx3005P qPCR apparatus (Agilent).

Every reaction had a pre-incubation to allow the reagents to come toreaction temperature and to test the effect that temperature had onamplification kinetics, enzyme performance, and signal fluorescence.

Amplification Procedure

The exact steps under which an amplification reaction was performed areas follows: 1) prepare master mix; 2) prepare primers with target or notarget; 3) add primer mixes to rows A-G of a 96 well plate, depending onnumber of reactions to be done per plate; 4) add master mix to row H ofthe same 96 well plate; 5) seal plate and do a pre-reaction incubationfor 15 seconds; 6) transfer master mix from row H to each primer mixrow; 7) seal and initiate preselected temperature profile and datacollection.

During the reaction, amplified product was measured at the end of everyexponential phase using a molecular beacon as described below. Thefluorescence of the molecular beacon in the reaction mixture wasmonitored to measure the amount of specific product being generatedduring a reaction which binds to the molecular beacon separating thefluorophore from the quencher, generating fluorescence.

Example 2: Results Using Unmodified Primers

To demonstrate the potential of this novel amplification technology,qSTAR was carried out using four replicates per target dilution across6-lags of genomic DNA input, and two replicates for no target controls(NTC). The results of experiments using unmodified primers (i.e. primermolecules not containing any chemically-modified, abnormal nucleic acidbases) are shown in FIG. 4 . The amount of signal (background subtractedfluorescence) for the “no-target control” is indicated by the dark line(“ntc”). The amount of signal generated in the presence of 20 cp, 200cp, 2 k, 20 k, 200 k, and 2M copies of target (genomic DNA of C.trachomatis) is indicated by the respective lines.

The qSTAR reactions display a linear coefficient of determination fromthe target input while also demonstrating an improvement in speed,sensitivity, and total fluorescence. It is surprising and unexpectedthat such an improvement and separation between target inputs could beachieved by controlling the temperature of the reactions between twoclose but distinctly different, temperature regions.

Without limiting the inventors to any particular theory, it is believedthat the amplification improvements can be attributed to at least twocharacteristics. In most nucleic acid amplification reactions, primerdimers eventually form, competing for limited reagents and, at lowtarget concentrations, primer dimers may potentially become the primaryamplification pathway for a reaction. Limiting or delaying the formationof primer dimers, even by a small amount, provides significant benefits.Because of the rapid nature of the amplification reaction, delayingprimer-dimer formation allows for preferred amplification pathways to befavoured (i.e. template generation) improving all aspects ofamplification. By initiating reactions at elevated temperatures thesetemplate pathways become favoured and even preferred. This is seen bythe improved sensitivity and speed in the gSTAR method, improvedfluorescence signal, tighter replicates and increased speed.

During the initiation phase, the reaction is run at an elevatedtemperature, 62° C. This elevated temperature selectively inhibits thenicking enzyme without permanently damaging it functionally (as shown inconjunction with amplification and FIG. 9B, described elsewhere). Duringthis initial phase, the polymerase is relatively favoured, allowing forrapid and specific extension, since the reaction temperature isrelatively close to the optimum temperature of the polymerase.

After the initiation phase of the reaction the temperature is reduced toa temperature which is closer to the optimum temperature for the nickingenzyme, resulting in increased efficiency and allowing for increasedgeneration of template. Since the desired template pathway has beenfavoured over errant pathways, specificity and sensitivity is greatlyincreased, which is further facilitated by qSTAR's temperature shuttlingand selective activity regulation of the enzymes.

The reaction mixture is continuously shuttled between 62 and 57° C., togive a controlled, rapid amplification technology that can be utilizedfor accurate quantitation.

The novel non-isothermal amplification method of the invention providesa substantial improvement over many types of existing amplificationreactions, including isothermal reactions and those that rely on hightemperatures for duplex dissociation. By controlling enzyme activity by“temperature gating” and optimizing reaction kinetics, the method of theinvention has improved consistency and control of amplification, whilstincreasing the sensitivity of detection, to allow for reliable andaccurate quantitation.

Example 3: Results Using 2′-O-Methyl Modified Primers

As described in U.S. Pat. Nos. 6,794,142 and 6,130,038, the use of2′-O-methyl modified primers is known to reduce primer dimer formationduring amplification. US 2005-0059003 describes the use of 2′-O-methylmodifications located at the 3′ end of SDA primers, suggesting that BstDNA Polymerase I and derivatives can efficiently utilize 2′-modifiedribonucleotides as primers for DNA synthesis. Target specific primerregions comprising one or more 2′ modified nucleotides (e.g.,2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2(methylamino)-2-oxoethyl], 2′-hydroxyl (RNA), 4′-thio,4′-CH3-O-2′-bridge, 4′-(CH3) 3-O-2′-bridge, 2′-LN A. and2′-O—(N-methylcarbamate, 2′-Suc-OH)) should improve amplificationreactions. The reactions were carried out using the enzyme-selectivetemperature shuttling (62-57° C.) as described in the preceding examplealong with a single 2′-O-methylated base or a string of 2′-O-methylatedbases located toward the 3′ of primers (illustrated schematically inFIG. 3 ).

The results of amplification using primers comprising one or more 2′modified nucleotides at the 3′ end are shown in FIGS. 5A-5C. Reactionswere carried out with a minimum of four replicates across all five loginput gDNA concentrations, along with no target control reactions. Asdemonstrated, the reactions are quantitative across a five-log rangewith a coefficient of determination greater than (data omitted forbrevity). The coefficient of determination was calculated by using asimilar method as described by Pfaffl in “A new mathematical model forrelative quantification in real-time RT-PCR”, (2001, Nucl. Acids Res. 29(9) e45). The starting point of the exponential phase, EP, ofamplification was determined by identifying where EP began abovebackground fluorescence. Background fluorescence was calculated byaveraging the first three reads of each reaction. The EP was thendetermined based on when the relative florescence for each reactionreached 2,000. Using the known input for each reaction the EP wasevaluated using a linear regression algorithm to determine thecoefficient of determination across log values. This standard curve wasgenerated and calculated for linearity, typically with qSTAR reactionsgenerating a R squared valued 0.99 or greater.

The data demonstrate (FIG. 5A) that the use of a primer incorporating asingle 2′-O-methylated nucleotide stalls amplification reactions,slowing the speed of the reactions for better resolution across allconcentrations of input. Further, the use of the qSTAR method not onlyimproved the use of 2′-O-methyl amplification, it illustrates thefunctionality of the method with known amplification modifications. Asshown in FIGS. 5B and 5C, incorporation of additional 2′-O-methylatedbases along the primer improves the separation of the amplification, orrise from baseline allowing for greater resolution, improving thequantitative ability of the technology. In essence, separation betweeneach concentration is improved by the slowing of the reactions caused byuse of 2′-O-methylated bases: for example, a reaction with a one cycleseparation in rise from baseline, when using unmodified primers, shows aseparation of 2 cycles when using primers incorporating the2′-O-methylated bases. This suggests that although 2′-O-methylmodifications do reduce the production of non-specific, errant,amplification in the exemplified method of the invention, the greaterbenefit of these modifications is to control the rate of reactions so asto permit greater resolution and more quantitative amplification.

Without limiting the applicants to any particular theory, the potentialimprovements obtained by using one or more 2′ modified nucleotides inthe primer region are hypothesized to be largely due to enhancements inthe initiation phase of amplification. During the initial extensionphase, two events help to explain the activity of 2′ modifiednucleotides in the amplification reaction of the invention. First,2′-O-methylated bases are known to lower the melting temperature ofDNA/DNP, duplexes resulting in more controlled initiation by tending toinhibit template template interactions thereby reducing the opportunityfor polymerase extension of nonspecific complexes formed by interactionsbetween primers. Secondly, it is possible that the polymerase stalls asthe nucleotide enters the binding pocket. In non-productive reactions(i.e., off-target or primer dimer formation), the stalling effect issufficient in minimizing aberrant extension because template binding isnear its melting temperature. Consequently, 2′ modifications are able torestrict undesirable amplification pathways because the reaction hasmired. qSTAR is able to leverage 2′ modifications and better regulatetarget amplification for tuning reactions for improved quantitativeability. This polymerase stalling further explains why qSTAR inconjunction with 2′-O-methyl modifications improve each other. Theinitial polymerase temperature region found in the exemplified method ofthe invention, besides decreasing primer dimer formation, slowsinitiation in a controlled and reliable manner. Furthermore, since qSTARrepeatedly shuttles to a lower temperature, the reduction in meltingtemperature caused by 2′ modifications can be curtailed as the reactionproceeds.

Example 4: Reproducibility

For validation of the qSTAR technology, a large replicate study wascarried out comparing qSTAR performance, against the performance of STARand other published isothermal conditions as described in U.S. Pat. No.9,562,263. Amplifications, (cISTAR vs. STAR vs. Isothermal), werecarried out using 100 plus replicates for reactions containing targetand 16 replicates for control reaction mixtures without target. Allconditions used the same buffers, polymerase, nicking enzyme and target.As shown in the scatter plot in FIG. 6 , qSTAR and STAR amplificationshows a clear improvement in average time (A_(T)) to achieveamplification to threshold level of fluorescence (T_(L)), improvedsensitivity, and a reduced standard deviation between replicates,compared to the isothermal amplification reaction. The A_(T) time forreactions performed according to the invention was 2.38 minutes, whilstthe A_(T) value for reactions performed according to conventionalisothermal protocols was 4.12 minutes, a difference which isstatistically significant (two-tailed test). (Note—failed reactions areshown as having an amplification time of 10 minutes—the maximum time forwhich reactions were run). Furthermore, the qSTAR method is animprovement over the STAR method with regards to speed. The qSTARtechnology demonstrated the tightest replicates, highest sensitivity,fastest amplification with the least number of outliers. Not to limitthe applicant to any particular theory, the significant reduction inamplification time is thought to be due to the improved initiation ofthe reaction, allowing for more efficient low copy amplification,minimized primer-dimer events, and increased specific product extensionwhich allows for faster product generation than previously disclosedmethods. Tighter replicates are achieved by leveraging the activity ofthe nicking enzyme and polymerase generating multiple chances forspecific, rapid, and controlled amplification of desired templates.

Example 5: qSTAR Functionality

A characteristic feature of the method of the present inventioncomprises the modulation of enzymatic activity by using smalltemperature changes during the amplification process, which temperaturechanges are far smaller than, say, the changes undergone duringperformance of qPCR. To verify that the nicking enzyme has reducedactivity during the initiation phase, yet that it is highly activeduring the exponential phase, the inventors have developed two uniqueprotein activity assays: a polymerase activity assay (“PAA”), and anicking activity assay (“NAA”).

Polymerase Activity Assay Design, Enzymes, and Oligonucleotides:

Synthetic oligonucleotides for the PAA were synthesized by IntegratedDNA Technologies (Coralville, IA). The design consists of threeoligonucleotides; the template oligo (NEF), (SEQ ID NO: 45′456-FAM/ACCGCGCGCACCGAGTCTGTCGGCAGCACCGCT-3′), priming oligo (PO),(SEQ ID NO: 5 5′-AGCGGTGCTGCCGACA-3′), and quenching oligo (POQ), (SEQID NO: 6 5′-GGTGCGCGCGGT/3BHQ_1/-3′). Together these threeoligonucleotides form a complex in solution each with unique functions,as shown in FIG. 7 . The NEF has a 5′ fluorophore, POQ has a 3′quenching moiety that absorbs the photons released by the 5′ templateoligo fluorophore. The PO serves as the initiation site for a stranddisplacement polymerase to extend and displace the quenching oligoallowing for fluorescence to be generated due to the quenching oligo nolonger being in proximity to the template oligo. Highly active stranddisplacing polymerases generate a fluorescent signal at an increasedrate compared to less active polymerases or those that lack standdisplacing activity.

Polymerase Activity Assay Conditions

The basic Polymerase Activity Assay (PAA) mixture contains a templateoligo (NEF) with a 5′-FAM modification, a priming oligo (PO) whichanneals to the template's 3-end, a quenching oligo (POQ) with a 3′-BHQ1modification which anneals to the template's 5′-end, and a polymeraseunder test (referenced above). The reactions were performed in a finalvolume of 25 μl, including 0.2 μM NEF, 0.3 μM PO, 0.7 μM POQ, and 1×PAAMaster Mix. At a 1× concentration, the PAA master mix contains thefollowing reagents; 12.5 mM MgSO4, 90 mM Tris-HCl (pH 8.5), 300 μM eachdNTPs, 15 mM NH₄CH₃CO₂, 15 mM Na₂SO₄, 5 mM DTT, 0.2 mg/ml BSA, 0.02%TRITON® X-100, 20 mM Rb₂SO₄, 10 mM L-Threonine, and 0.03 U/μlpolymerase. The reactions are run isothermally to determine the activityof selected enzymes at specific temperatures. The PAA was performed withthe Agilent Mx3005P qPCR apparatus (Agilent). Every reaction had apre-reaction incubation to allow the reagents to come to temperature totest the effect of the selected temperature and prevent any variation asreactions heated up. Each reaction assessed amplification kinetics,enzyme performance, and signal fluorescence.

Nicking Activity Assay (NAA) Design, Enzymes, and Oligonucleotides:

Synthetic oligonucleotides for the NAA were synthesized by IntegratedDNA Technologies (Coralville, IA). The assay involves twooligonucleotides; the template oligo (NEQ), (SEQ ID NO: 75′-ACCGCGCGCACCGAGTCTGTCGGCA/3BHQ_1/-3′) and priming oligo (POF, SEQ IDNO: 8 5′-/56-FAM/CTGCCGACAGACTCGGTGCGCGCGGT-3′). Together theseoligonucleotides form a complex in solution each with unique functions,as shown in FIG. 8 . The template oligo has a nicking site for nickingendonuclease activity and downstream a 3′ quencher. The priming oligohas the complementary nicking site sequence and a 5′ fluorophore. Whenin solution the two form a complex that completes a nicking binding siteallowing for the nicking endonuclease to cut. The oligonucleotidequencher 3′ of the nick site, following a nick by a nickingendonuclease, now has a low melting temperature. Because the reaction isperformed above this melting temperature, the shortened fragmentcontaining the quencher is released from the complex, resulting inunquenched fluorescence. The more active the nicking enzyme the fasterand greater the florescent signal is generated.

Nicking Activity Assay Conditions

The basic NAA mixture contains the template oligo (NEQ) with a 3′-BHQ1modification, and the priming oligo (POF) with a 5′-FAM modificationwhich anneals to the template, and a nicking endonuclease to be tested.The reactions were performed in a final volume of 25 μl, including 1.3μM NEQ, 1.6 μM POF, and 1×NAA Master Mix. At a 1× concentration, the NAAmaster mix contains the following reagents; 12.5 mM MgSO₄, 90 mMTris-HCl (pH 8.5), 15 mM NH₄CH₃CO₂, 15 mM Na₂SO₄, 5 mM DTT, 0.2 mg/mlBSA, 0.02% TRITON® X-100, 20 mM Rb₂SO₄, 10 mM L-threonine, and 0.008U/μl nicking endonuclease. The reactions are run isothermally todetermine the activity of selected enzymes at specific temperatures. TheNAA was performed with the Agilent Mx3005P qPCR apparatus (Agilent).Every reaction had a pre-reaction incubation to allow the reagents tocome to temperature to test the effect of the selected temperature andprevent any variation as reactions heated up. Each reaction assessedamplification kinetics, enzyme performance, and signal fluorescence.

Temperature Profile of Enzymes

FIG. 9A shows the polymerase activity assay for six isothermalconditions. At 63° C. the polymerase has the strongest activity andkinetics, as determined by the slope of the fluorescent curve and totalfluorescence. Each subsequent drop in temperature, 60° C., 55° C., 50°C., and 45° C. shows a decrease in activity until arriving at 40° C. Atthis low temperature, the activity of the polymerase appears to besubstantially non-existent.

FIG. 9B shows the nicking activity assay for six isothermal conditions.Unlike the polymerase assay, which shows a clear optimal temperaturetowards the top end of the preferred range of temperatures for the qSTARmethod, the nicking activity assay shows an optimum (about 55° C.)towards the lower end of the preferred range of temperatures for theqSTAR method, while demonstrating little to no activity at 63° C. Allother temperatures show some level of activity for the nicking enzyme.

The data from these assays demonstrate the distinctive nature of theqSTAR technology. Unlike other amplification methods that rely on stranddisplacement and/or temperature separation, qSTAR uniquely uses“temperature gating” to modulate enzyme activity and control rapidamplification. Recognizing the unique features of these enzymes andtemperature dependence upon activity, the inventors have developed a newrapid, specific, controlled amplification technology that can quantitateunknown sample inputs in under six minutes.

Without being bound by any particular theory, it is believed that inthis example qSTAR involves activity modulation of the nicking enzyme asit amplifies between two temperatures. 63 C.° and 57 C.° are thepreferred temperature choice in the exemplified system described above(based upon current protein activity profiles) because they allow forcontrolled amplification, a requirement for any quantitative technology.It is further believed that controlling the activity of either enzyme isdesirable to manage a known efficient amplification event forquantitation of unknown nucleic acid material.

Example 6: qSTAR Amplification Results Using qPCR Polymerases

To demonstrate the unexpected properties of qSTAR versus otheramplification technologies, such as PCR, a comparison of common PCRpolymerases was performed, showing that common PCR polymerases andmethods are inactive in the qSTAR method. Four PCR polymerases; VENT™,DEEP VENT™, TAQ, AND PHUSION® were used for amplification in a qPCRmethod, as described below, and compared with the qSTAR method. Becausemolecular beacons only measure an increase in the total amount ofspecific single-stranded DNA product, non-specific amplification productis not measured independently of the intended amplification product. Tomeasure the production of all amplification products (e.g. includingthose arising from primer dimer formation), reactions were carried outin the presence of SYBR® Green I. SYBR® Green I is one of the mostsensitive dyes known for detecting single-stranded DNA, RNA, anddouble-stranded DNA. Because SYBR® Green I has a low intrinsicfluorescence, it is a good choice for detection of total amplificationin a reaction, both specific and non-specific, to demonstrate thatcommon PCR polymerases are inactive in the qSTAR method.

qPCR/qSTAR Assay Design, Master Mix, and Oligonucleotides:

Synthetic oligonucleotides for the in-house qPCR assay (Ctx) weresynthesized by Integrated DNA Technologies (Coralville, IA) and designedfor the amplification of Chlamydia Trachomatis genomic DNA. The designconsists of two oligonucleotides; the forward priming oligo (Ctx_L.F1,SEQ ID NO: 9 AAAAAGATTTCCCCGAATTAG), and a reverse priming oligo(Ctx_L.R1_3′(-2), SEQ ID NO: 10 AGTTACTTTTTCCTTGTTT). Oligonucleotideswere synthesized by Integrated DNA Technologies (Coralville, IA). SYBRGreen I Nucleic Acid Stain (Lonza Rockland, Inc. P/N 50513) was used asan intercalating dye for detection of double stranded DNA (dsDNA)products. PCR master mix and polymerases used were from New EnglandBiolabs (Ipswich, MA); 10× THERMOPOL® Reaction Buffer, VENT™ (exo-) DNAPolymerase (P/N M0257S), DEEP VENT™ (exo-) (P/N M0257S), and Taq DNAPolymerase (P/N M0267S), 5× PHUSION® HF Buffer, and PHUSION® HF DNAPolymerase (P/N M0530S). Genomic DNA for Chlamydia Trachomatis (Strain:UW-36/Cx) (P/N VR-886D) was purchased through ATCC (Manassas, VA).

qPCR/qSTAR Assay Conditions

The basic in-house qPCR assay (Ctx) mixture contained a forward primeroligo, a reverse primer oligo, a dsDNA intercalating dye, a knownconcentration of genomic DNA template, a 1× concentration of commercialPCR master mix, and its corresponding polymerase (mentioned above). Thereactions were performed in a final volume of 25 μl, including 0.3 μMF1, 0.3 μM R1, 0.1× SYBR® Green I, 1× commercial PCR Master Mix, 0.03U/μl polymerase, and 5,000 copies of genomic DNA template.

The in-house qPCR assay was run using 2 methods; a temperature profilereplicating qSTAR technology or that of conventional qPCR. In the qSTARmethod, the temperature of the reactions was controlled between twodiscreet temperatures to take advantage of enzyme activities. Theinitiation phase, substantially (polymerase only activity), was at theelevated temperature of 62° C. for two seconds. The exponential phase,(polymerase and nicking enzyme activity), was closer to the optimaltemperature for the nicking enzyme's activity at 57° C. for fiveseconds. The total time for a complete shuttle was 15 seconds, which ismore than double the dwell times at the maximum and minimum temperaturedue to the limits of the apparatus in changing temperature. The qPCRreactions were preformed using a 2-step program; 95° C. for fifteenseconds followed by 60° C. for sixty seconds, cycle 50× times.Amplification and qSTAR product detection were performed with theAgilent Mx3005P qPCR apparatus (Agilent).

Results

In FIGS. 10A-B show the real time data for qPCR amplification of fivethousand copies of genomic Chlamydia trachomatis DNA compared to NoTarget control (“ntc”). Clearly seen is the amplification or activity ofall polymerases using this qPCR method. It should also be noted thatthree out of the four polymerases show activity in the no targetconditions, which is probably due to primer dimer formation. If theqSTAR method were similar to qPCR or previously reported thermal cyclingamplification technologies, one would expect all or at a minimum one ofthese polymerases being active using the qSTAR method.

In FIGS. 11A-11D, the real time data demonstrate the inability of allfour of the aforementioned polymerases to show any activity in reactionswith no target or using 10, 100, 1K, 10K copies of genomic Chlamydiatrachomatis DNA under the qSTAR temperature shuttling protocol. It issurprising that not one of these polymerases, all being used in theiroptimal temperature ranges, is able to show even a small amount ofactivity during the course of the incubation. Not to limit the inventorsto any particular theory, this is believed to be due to following; (a)qSTAR conditions require strand displacement polymerases working inconjunction with nicking enzymes; without this combination of enzymes,amplification cannot proceed because product turnover is unable toprogress; and (b) PCR and other cycling methods rely on elevatedtemperature (˜95° C.) to strand-separate amplicons for amplificationprogression; since the qSTAR method does not use such an elevatedtemperature and instead uses more moderate temperature shuttling forcontrolling enzyme activity (rather than for strand separation), itcould help explain the inability of any of these enzymes to show anyactivity in the qSTAR protocol conditions.

Example 7: qSTAR Versus qPCR Results

To demonstrate the quantitative nature of qSTAR, a comparison wasperformed versus qPCR. If qSTAR is quantitative one would expect thetechnology to have a high coefficient of determination, and be able tocorrectly predict the amount of genomic DNA in blinded samples ascompared to qPCR.

C. trachomatis qPCR Assay Design, Master Mix, and Oligonucleotides:

Synthetic oligonucleotides (1) for the C. trachomatis qPCR assay (CtP)were designed for the amplification of Chlamydia Trachomatis genomicDNA. The assay involves the use of three oligonucleotides; a forwardpriming oligo, a reverse priming oligo, and a dual-labelled probe.Oligonucleotides were synthesized by Integrated DNA Technologies(Coralville, IA). The PCR master mix used, PRIMETIME® Gene ExpressionMaster Mix (P/N 1055770), was purchased from Integrated DNA Technologies(Coralville, IA). Genomic DNA for Chlamydia Trachomatis (Strain:UW-36/Cx) (P/N VR-886D) was purchased through ATCC (Manassas, VA).

C. trachomatis qPCR Assay Conditions

The basic qPCR assay (CtP) mixture contained two primers, polymerase andgenomic DNA. The reactions were performed in a final volume of 25 μl,including 0.3 μM forward primer, 0.3 μM reverse primer, 0.1 μMdual-labelled probe, 1× commercial PCR Master Mix, and variousconcentrations of genomic DNA template starting from 100,000 copies.Standard curves were generated using 10-fold dilutions of the genomicDNA. The gSTAR was performed as previously described along with theabove standard curves. The qPCR reactions were performed using a 2-stepprogram; 95° C. for fifteen seconds followed by 60° C. for sixtyseconds, cycle 50× times.

Results

FIG. 12 shows the qPCR real time data for the standard curve and 5unknown samples. The coefficient of determination for the standard curvewas 0.9984, across a 5 log range. qPCR was able to correctly call allfive unknown samples. FIG. 13 shows the qSTAR real time data for thestandard curve and five unknown samples. The coefficient ofdetermination for the standard curve was 0.9981, across a 6 log range.qSTAR was able to correctly call all five unknown samples. Table 2 showsa summary comparison of the two technologies, it is clear from thesummary that qSTAR is comparable to qPCR when using the technology forquantitation.

TABLE 2 qPCR Estimated qSTAR Estimated Sample Copy # Added Copy # Copy #UNK01 250 114 276 UNK02 50000 45848 75699 UNK03 0 0 0 UNK04 100000 96886140611 UNK05 8000 5816 10630

Example 8: gSTAR Elevated Temperature Ranges

A further benefit of qSTAR technology is the ability to amplify acrossvarious temperature ranges. As described in U.S. Pat. Nos. 5,712,124,9,562,263, 5,399,391, and 6,814,943, most technologies have a tighttemperature range in which amplification can occur, and deviating fromthese ranges inhibits the reaction. To demonstrate the versatility ofqSTAR, amplifications were carried out as described in Table 3 below.

TABLE 3 qSTAR Conditions Initiation Phase Time Exponential Phase Time63° C. 1 second 57° C. 5 seconds 64° C. 1 second 57° C. 5 seconds 65° C.1 second 57° C. 5 seconds 66° C. 1 second 57° C. 5 seconds

FIGS. 14A, US, 14C, and 14D are graphs of fluorescence (arbitrary units)against time (minutes). FIG. 14A shows the results for reactionsstarting at 63° C. FIG. 14B shows the results for reactions starting at64° C. FIG. 14C shows the results for reactions starting at 65° C. andFIG. 14D shows the results for reactions starting at 66° C. In allcases, “no target” negative control reactions did not generate anyfluorescence signal, whereas there was good amplification for 10, 100,and 1,000 copies. Although the fluorescence signal was slightly higherfor the 63° C. reaction, all temperature conditions demonstrated strongamplification in less than 3 minutes. As long as enzyme modulation isachieved the gSTAR method can amplify wed. It is believed that anytemperature above 62° C. significantly reduces nicking enzyme activity(in respect of the exemplified nicking enzyme, Nt. Bst NBI).

Example 9: gSTAR Outside Known Temperature Ranges

Quantitative Polymerase Chain Reaction (qPCR) as described in U.S. Pat.No. 6,814,943 describes temperature ranges for thermal cycling.Typically for qPCR the following procedure is undertaken: denaturationaround 95° C., annealing around 55° C., extension around 70° C. It wouldbe surprising and unexpected if a technology could amplify in distinctlydifferent temperature regions. Furthermore, individuals with knowledgein the art would not expect such a large temperature window for atechnology to work in. WO 2011/030145A1 describes “wobbling” in whichthe assay temperature oscillates around a published isothermaltemperature setpoint of no more than 15° C., but more preferably around5° C. This temperature “oscillation” for some isothermal technologieshas allowed for improved amplification kinetics. It would be surprisingif qSTAR is able to work in dramatically different temperature rangesand still achieve amplification.

Amplification Conditions

The low temperature gSTAR mixture contained two primers (SEQ ID NO: 11(5′-tGACTCCAcAcGGAGTCataaATCCTGCTGCmUA-3) and SEQ ID NO: 12(5′-polymerase supplied by ArticZymes (Tromso, Norway), and nickingenzyme (referenced previously). The reactions were performed in a finalvolume of 25 μl, including 1.0 μM of the forward primer, 0.5 μM of thereverse primer, 0.25 μM molecular beacon (SEQ ID NO: 13(5′-/56-FAM/tgaggTGCTGCTATGCCTCA/3IABkFQ/-3′)), 10 μl gSTAR Master Mixand 5 μl DNA sample. gSTAR master mix contained the following reagents;12.5 mM MgSO₄, 90 mM Tris-HCl (pH 8.5), 300 μM each dNTPs, 20 mM NH₄OAc,30 mM NaOAc, 2 mM DTT, 0.02% TRITON® X-100, 12.5U nicking endonuclease,75U polymerase. The temperature of the reactions was controlled betweentwo discreet temperature phases to take advantage of inherent enzymeactivities. The exponential phase, consisting primarily of polymeraseand nicking activity, was at the elevated temperature of 45° C. for twoseconds. The initiation phase, in which the polymerase is highly activeand nicking enzyme has greatly reduced activity, was held at 38° C. forfive seconds. The total time for a complete shuttle was 15 seconds,which is more than double the dwell times at each of the maximum andminimum temperatures due to the limits of the apparatus in changingtemperature. Amplification and gSTAR product detection were performedwith the Agilent Mx3005P gPCR apparatus (Agilent),

Results

FIG. 15 shows real time quantitative data for qSTAR amplifying in theabove referenced range. The first thing that should be noticed is that,in this example, compared to the preceding examples the temperaturephases have been switched: the higher temperature phase is forexponential amplification, in which both enzymes are active, while thelower temperature is for initiation, in which the polymerase is highlyactive and the nicking enzyme is relatively inhibited. The temperaturedifference between qSTAR and such “low temperature” qSTAR is 24° C. Itis surprising and unexpected that a technology can work over such alarge range of temperatures and further demonstrates that thisamplification method is unlike any amplification method knownpreviously, to the best knowledge of the inventors.

Not to limit the inventors to any particular theory, it is believed thatqSTAR is able still to achieve amplification at these low temperaturesbecause the nicking enzyme activity is greatly reduced at the lowertemperature. This gating of enzymes allows for controlled and preciseamplification of templates and the inventors can envisage many ways inwhich multiple enzymes, primers, and temperature schemes can be used ina single reaction to achieve new, fast, and quantitative results.

Example 10: Results Using Ribonucleic Acid

qSTAR can amplify from any nucleic acid, using any composition of DNA(cDNA and gDNA), RNA (mRNA, tRNA, rRNA, siRNA, microRNA), RNA/DNAanalogs, sugar analogs, hybrids, polyamide nucleic acid, and other knownanalogs. Amplification of ribosomal RNA was carried out as describedbelow.

Enzymes, Oligonucleotides, and Target:

Listeria monocytogenes was used as the target for the development of aqSTAR RNA assay. Listeria monocytogenes (ATCC VR-886) genomic DNA wasacquired from American Type Culture Collection (Manassas, VA). Initialscreening was performed on gDNA, and a 23S region of ribosomal RNA wasfound to be amplified with primers LMONF72 ACAC 5-OM (SEQ ID NO: 14,5′-GGACTCGACACCGAGTCCAGTTACGATTmTmGmTmTmG-3′) and LMONR86 ATAT (SEQ IDNO: 15, 5′-gGACTCCATATGGAGTCCTACGGCTCCGCTTTT-3′). The resulting DNAtemplate was detected using a molecular beacon LMONMB1 (SEQ ID NO: 16,5′-FAM/gctgcGTTCCAATTCGCCTTTTTCGCagc/BHQ1-3′) as described in EP No.0728218.

Total RNA was isolated using the RNEASY® Plus mini kit Qiagen (Hilden,Germany) combined with rapid mechanical lysis on a Mini Bead Mill 4(VWR). Listeria monocytogenes (ATCC BAA-2660) was acquired from AmericanType Culture Collection (Manassas, VA), and revived by plating onbrain-heart infusion agar plates (BHI). A single colony was used toinoculate 25 mL of BHI media that was grown for 18 hours at 37° C. toreach stationary phase. The culture was then back-diluted into two 50 mLportions of BHI in 250 mL flasks and grown for an additional four hoursprior to harvest. Bacteria were harvested from two 30 mL aliquots of theback-diluted culture at for 15 min. The pellets were resuspended andcombined into 5 mL of RNALATER™ RNA stabilization Reagent (Qiagen) andallowed to incubate for 10 min at room temperature. The bacteria wereharvested and resuspended in 5 mL of RLT lysis buffer Bacteria, andhomogenised on the Mini Bead Mill (VWR) at setting 5 (3×30 seconds withone minute on ice between pulses).

Total RNA was purified per manufacturer's directions (Qiagen). GenomicDNA was removed by passing lysates over a DNA-binding column provided inthe RNEASY® Plus purification kit. Genomic DNA contamination was furtherreduced by utilizing an on-column RNase free DNase I (Qiagen) digestionof samples on the RNEASY® RNA-binding column. Bst X DNA Polymerase waspurchased from Beverly Qiagen (Beverly, MA). OMNISCRIPT®, a ReverseTranscriptase, was purchased from Qiagen (Hilden, Germany). Nt.BstNBInicking endonuclease was purchased from New England BioLabs (Ipswich,MA) as described in U.S. Pat. No. 6,191,267. Oligonucleotides andmolecular beacons were synthesized by Integrated DNA Technologies(Coralville, IA).

Amplification Conditions:

The basic gSTAR mixture contained everything as described in example 1above with the additional inclusion of the following: 4U of ReverseTranscriptase (referenced above).

Results

The results are shown in FIG. 16 which is a graph of fluorescence(arbitrary units) against time (minutes). Negative control reactions didnot generate any fluorescence signal, whereas 100, 1,000, 10,000,100,000, 1,000,000 copy number target reactions generated fluorescencesignal above threshold. The results show that gSTAR can amplifyeffectively from a reverse transcribed RNA target. Furthermore the dataindicates it could be used to quantitate unknown RNA sample inputs.

1. A method of performing a non-isothermal nucleic acid amplificationreaction, the method comprising the steps of: (a) mixing a targetsequence with one or more complementary single stranded primers inconditions which permit a hybridisation event in which the primershybridise to the target, which hybridisation event, directly orindirectly, leads to the formation of a duplex structure comprising twonicking sites disposed at or near opposite ends of the duplex; andperforming an amplification process by; (b) using a nicking enzyme tocause a nick at each of said nicking sites in the strands of the duplex;(c) using a polymerase to extend the nicked strands to as to form newlysynthesised nucleic acid, which extension with the polymerase recreatesnicking sites; (d) repeating steps (b) and (c) as desired so as to causethe production of multiple copies of the newly synthesised nucleic acid;characterised in that the temperature at which the method is performedis non-isothermal, and subject to shuttling, a plurality of times,between an upper temperature and a lower temperature during theamplification process of steps (b)-(d), wherein at the uppertemperature, one of said polymerase or nicking enzyme is more activethan the other of said enzymes, such that there is a disparity in theactivity of the enzymes, and at the lower temperature the disparity inthe activity of the enzymes is reduced or reversed; wherein thetemperature of the reaction mixture is held constant at the uppertemperature for upper temperature dwell time and/or the reaction mixtureis held constant at the lower temperature for a lower temperature dwelltime. 2-31. (canceled)
 32. The method according to claim 1, wherein theupper temperature dwell time and the lower temperature dwell time arethe same.
 33. The method according to claim 1, wherein the uppertemperature dwell time and the lower temperature dwell time aredifferent.
 34. The method according to claim 32, wherein the uppertemperature dwell time is less then the lower temperature dwell time.35. The method according to claim 32, wherein the upper temperaturedwell time is greater than the lower temperature dwell time.
 36. Themethod according to claim 1, wherein the upper temperature dwell timeand/or the lower temperature dwell time are varied during the pluralityof temperature shuttles.
 37. The method according to claim 1, whereinthe transition times between the upper temperature and the lowertemperature and between the lower temperature and the upper temperatureare substantially the same for the plurality of temperature shuttles.38. The method according to claim 1, wherein the transition time betweenthe upper temperature and the lower temperature is varied across theplurality of temperature shuttles.
 39. The method according to claim 1,wherein the transition time between the lower temperature and the uppertemperature is varied across the plurality of temperature shuttles. 40.The method according to claim 1, wherein the upper temperature is in therange 50-68° C.
 41. The method according to claim 1, wherein the lowertemperature is in the range 20.0-58.5° C.
 42. The method according toclaim 1, wherein the temperature shuttling is performed continuously fora plurality of shuttles and over a period of at least two minutes. 43.The method according to claim 1, wherein each of the plurality oftemperature shuttles has a duration in the range 5-60 seconds.
 44. Themethod according to claim 1, wherein each of the plurality oftemperature shuttles has a dwell time at the upper temperature in therange 1-10 seconds.
 45. The method according to claim 1, wherein each ofthe plurality of temperature shuttles has a dwell time at the lowertemperature in the range 2-40 seconds.
 46. The method according to claim1, wherein each of the plurality of temperature shuttles has atransition time between the lower temperature and the upper temperaturein the range seconds.
 47. A method of determining the amount and/orconcentration of a target polynucleotide in a sample, the methodcomprising the steps of: performing the amplification reaction of claim1 to amplify the target polynucleotide in the sample; and detecting, ina quantitative manner, the direct or indirect product(s) of theamplification reaction, so as to allow a determination of the amountand/or concentration of the target polynucleotide in the sample.